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Introduction
Embryonic stem cells are capable of self-renewing and differentiating to the desired fate depending on its position within the body. Stem cell homeostasis is maintained through epigenetic mechanisms that are highly dynamic in regulating the chromatin structure as well as specific gene transcription programs [1]. Epigenetics has been used to refer to changes in gene expression, which are heritable through modifications not affecting the DNA sequence.

The mammalian epigenome undergoes global remodeling during early stem cell development that requires commitment of cells to be restricted to the desired lineage. There has been multiple evidence suggesting that the maintenance of the lineage commitment of stem cells are controlled by epigenetic mechanisms such as DNA methylation, histone modifications and regulation of ATP-dependent remolding of chromatin structure [1]. Based on the histone code hypothesis, distinct covalent histone modifications can lead to functionally distinct chromatin structures that influence the fate of the cell.

This regulation of chromatin through epigenetic modifications is a molecular mechanism that will determine whether the cell will continue to differentiate into the desired fate. A research study performed by Lee et al. examined the affects of epigenetic modifications on the chromatin structure and the modulation of these epigenetic markers during stem cell differentiation through in vitro differentiation of murine embryonic stem (ES) cells [2].

Experimental background
Embryonic stem cells exhibit dramatic and complex alterations to both global and site-specific chromatin structures. Lee et al. performed an experiment to determine the importance of deacetylation and acetylation for stem cell differentiation by looking at global acetylation and methylation levels at certain site-specific modification in histones H3K9 and H3K4. Gene expression at these histones regulated by epigenetic modifications is critical in restricting the embryonic stem cell to desired cell lineages and developing cellular memory.

For mammalian cells, the maintenance of cytosine methylation is catalyzed by DNA methyltransferases and any disruption to these methyltransferases will cause a lethal phenotype to the embryo. Cytosine methylation is examined at H3K9, which is associated with inactive heterochromatin and occurs mainly at CpG dinucleotides while global acetylation is examined at H3K4, which is associated with active euchromatin. The mammalian zygotic genome undergoes active and passive global cytosine demethylation following fertilization that reaches a minimal point of 20% CpG methylation at the blastocyst stage to which is then followed by a wave of methylation that reprograms the chromatin structure in order to restore global levels of CpG methylation to 60% [2]. Embryonic stem cells containing reduced or elevation levels of methylation are viable but unable to differentiate and therefore requires critical regulation of cytosine methylation for mammalian development.

Effects of global histone modifications during Embryonic Stem Cell differentiation
Histones modifications in chromatin were analyzed at various time intervals (along a 6 day period) following the initiation of in vitro embryonic stem cell differentiation. Differentiation was triggered by the removal of Leukemia Inhibitory Factor (LIF) which inhibits differentiation. Representative data of the histone modifications at the specific sites were assessed using Western blotting. The data confirms that strong deacetylation at the H3K4 and H3K9 positions of histone H3 one day after LIF removal, followed by a small increase in acetylation by day 2.

The histone H3K4 methylation also decreased after 1 day of LIF removal but showed a rebound between days 2-4 of differentiation finally ending with a decrease in methylation on day 5. These results indicate a decrease in the level of active euchromatin epigenetic marks upon initiation of embryonic stem cell differentiation to which is then followed immediately by reprogramming of the epigenome. Histone modifications of H3K9 show a decrease in di- and tri-methylation of undifferentiated embryonic stem cells and had a gradual increase in methylation during the 6-day time course of in vitro differentiation, which indicated that there is a global increase of inactive heterochromatin levels at his histone mark.

As the embryonic stem cell undergoes differentiation the markers for active euchromatin (histone acetylation and H3K4 methylation) are decreased after the removal of LIF showing that the cell is indeed becoming more differentiated. The slight rebound in each of these marks allows for further differentiation to occur by allowing another opportunity to decrease the markers once again bring the cell closer to its desired fate. Since there is also an increase throughout the 6 day period in H3K9me, a marker for active heterochromatin, once differentiation occurs it is concluded that the formation of heterochromatin occurs as the cell is differentiated into its desired fate making the cell inactive to prevent further differentiation.

DNA methylation in differentiated v. undifferentiated cells:

Global levels of cytosine methylation were compared between undifferentiated and differentiated embryonic stem cells. Global 5-methylcytosine levels have been quantitated prior to differentiation and after in vitro differentiation. The global cytosine methylation pattern appears to be established prior to the reprogramming of the histone code that occurs upon in vitro differentiation of embryonic stem cells.

As the embryonic stem cell undergoes differentiation the level of DNA methylation increases. This is in agreement with findings that show that there is an increase in inactive heterochromatin during differentiation.

Supplemental effects of Methylation with DNMTs
In mammals, DNA methylation plays a role in regulating a key component of multipotency-the ability to rapidly self-renew. Khavari et al. discussed the fundamental mechanisms of DNA methylation and the interaction with several pathways regulating differentiation [9]. New approaches studying the genomic status of DNA methylation in various states of differentiation have shown that methylation at CpG sites associated with putative enhancers are important in this process. DNA methylation can modulate the binding affinities of transcription factors by recruiting repressors such as MeCP2 which display binding specificity for sequences containing methylated CpG dinucleotides. DNA methylation is controlled by certain methyltransferases, DMNTs, which perform different functions depending on each one. DNMT3A and DNMT3B have both been linked to play a role in the establishment of DNA methylation pattern in the early development of the stem cell, whereas DNMT1 plays a role that is required to methylate a newly synthesized strand of DNA after the cell has undergone replication in order to sustain the epigenetic regulatory state. Numerous proteins can physically interact with DNMTs themselves, which help target DNMT1 to hemi-methylated DNA.

Several new studies point to the central role of DNA methylation interacting with the regulation of cell cycles and DNA repair pathways in order to maintain the undifferentiated state. In embryonic stem cells, DNMT1 depletion within the undifferentiated progenitor cell compartment led to cell cycle arrest, premature differentiation and a failure of tissue self-renewal. The loss of DNMT1 occurred from profound effects associated with activation of differentiation genes and loss of genes promoting cell cycle progression, thus indicating that DNMT1 and other DNMTs do not continuously suppress differentiation and thus maintain the pluripotent state.

These studies point to the important of the interaction of DNMTs in order to maintain stem cell states allowing for further differentiation and formation of heterochromatin to occur.

Epigenetic modifications of regulated genes during ESC differentiation
Okamoto et al. [3] previously documented the expression level of the Oct4 gene decreasing with embryonic stem cell differentiation. Lee et al performed a ChIP analysis of the Oct4 promoter, associated with undifferentiated cells, region to examine the epigenetic modifications of regulated genes undergoing development during embryonic stem cell differentiation. This promoter region decreased at H3K4 methylation and H3K9 acetylation sites and increased at the H3K9 methylation site during differentiation. Analysis of a CpG motif of the Oct4 gene promoter revealed a progressive increase of DNA methylation and was completely methylated at day 10 of differentiation as previously reported in Gidekel and Bergman [4].

These results indicate that there was a shift from the active eurchromatin to the inactive heterochromatin due to the decrease of acetylation/H3K4 and an increase of H3K9me. This means that the cell is becoming differentiated at the Oct4 gene, which is coincident with the silence of Oct4 gene expression.

Another site specific gene tested for histone modification was a Brachyury gene, a marker of mesoderm differentiation and is only slightly expressed in undifferentiated embryonic stem cells. "Brachyury" was induced at day 5 of differentiation and completely silencing by day 10, corresponding to the last day of differentiation [5]. The ChIP analysis of the "Brachyury" gene promoter revealed increase of expression in mono- and di-methylation of H3K4 at day 0 and 5 of embryonic stem cell differentiation with a loss of gene expression at day 10. H3K4 trimethylation coincides with the time of highest Brachyury gene expression since it only had gene expression on day 5. H3K4 methylations in all forms are absent at day 10 of differentiation, which correlates with the silencing of Brachyury gene expression. Mono-methylation of both histones produced expression at day 0 indication a marker that is not useful for chromatin structure. Acetlyation of H3K9 does not correlate to Brachyury gene expression since it was down regulated at the induction of differentiation. Upon examining of DNA methylation expression, there was no formation of intermediate sized bad in the Southern analysis suggesting that CpG motifs upstream of the promoter region are not methylated in the absence of cytosine methylation at this site. It is demonstrated from these studies that both H3K9 di-and tri-methylation correlate with the DNA methylation and gene expression while H3K4 tri-methylation is associated the highest gene expression stage of the Brachyury gene. A previous report from Santos-Rosa [6] is in agreement with these data showing that active genes are associated with H3K4 tri-methylation in yeast.

This data indicated the same results with the Oct4 gene in that heterochromatin is forming at differentiation occurs again coinciding with the silence of Brachyury gene expression.

Effect of TSA on the Stem Cell Differentiation
Leukemia initiation factor (LIF) was removed from all the cell lines. LIF inhibits the cell differentiation and its removal allows the cell lines to go through the cell differentiation. The cell lines were treated with Trichostatin A (TSA) - the histone deacetylase inhibitor for 6 days. One group (10nM TSA) of cell lines was treated with 10nM of TSA. The western analysis showed the lack of initial deacetylation on Day-1 which, was observed in the control for the embryonic stem cell differentiation. The lack of histone deacetylase activity allowed the acetylation of H3K9 and H4. ES cells were also analyzed morphologically to observe the formation of embryoid body formation as one of the measures of cell differentiation. The 10nM TSA treated cells failed to form the embryoid body by Day-6 as observed in the control cell line. This implies that the ES cells treated with TSA lacked the deacetylation on Day-1 and failed to differentiate after the removal of LIF. Second group,’-TSA Day4’ was treated with TSA for 3days. As soon as the TSA treatment was stopped, on Day-4 the deacetylation was observed and the acetylation recovered on Day-5. The morphological examination showed the formation of embryoid body formation by Day-6. In addition, “Interestingly” the embryoid body formation was faster than the control cell line. This suggests that the ‘-TSA Day4’ lines were responding to the removal of LIF but, were unable to acquire any differentiation phenotype. They were able to acquire the differentiation phenotype after the cessation of TSA treatment and at rapid rate. The morphological examination of the third group,’ 5 nM TSA’ showed the intermediate effect between the control and 10nM-TSA group. The lower dose of TSA allowed the formation of some embryoid body formation. This experiment shows that TSA inhibits HDAC and the activity of histone deacetylase is required for the embryonic stem cell differentiation. Without the initial deacetylation on Day-1, the ES cells cannot go through the differentiation.

Alkaline Phosphatase Activity-another measure of ES cell differentiation
In the control cell line, the activity of alkaline phosphatase activity is lowered upon differentiation. This implies that the extinction of alkaline phosphatase is high. In ‘10nM TSA’ group, TSA failed to lower the activity of alkaline phosphatase activity and low extinction of alkaline phosphatase on Day-6, suggesting that ES cell differentiation was not observed. The significant increase in the alkaline phosphatase extinction was observed in ‘-TSA Day4’ group suggesting the occurrence of ES cell differentiation. The examination of alkaline phosphatase activity strengthens the observations from the morphological examination and reemphasizes that the initial deacetylation of histone is required for the ES cell differentiation.

HDAC1, but not HDAC2 controls the Embryonic Stem Cell Differentiation
Dovery et al. (2010) used HDAC knockout mice to demonstrate whether HDAC1 or HDAC2 was important for the ES cell differentiation.Examination of global histone acetylation in the absence of HDAC 1 showed the increase in the acetylation status. The global histone acetylation levels were unchanged by the loss of HDAC2. In order to analyze the process of HDAC knockout mouse in detail, the knockout mice ES cells were used to generate embryoid bodies. It showed that just before or during gastrulation, ES cells lacking HDAC1 acquired phenotypically apparent developmental defects. The continued culture of HDAC1 knockout ES cells showed that the embryoid bodies formed became irregular and reduced in size rather than uniformly spherical shape in the control. ES cell proliferation was unaffected by the loss of either HDAC1 or HDAC2 but the differentiation of ES cells were affected with that lack of HDAC 1. Hence,The study of HDAC1 and HDAC2 in stem cell identified the requirement of HDAC1 in the cell fate determination during differentiation [7].

The Future
Any disturbance of a stable epigenetic regulation of gene expression mediated by DNA methylation is associated with a number of human disorders, including cancer as well as congenital diseases such as pseudohypoparathyroidism type IA, Beckwith- Wiedemann, Prader-Willi and Angelman syndromes, which are each caused by altered methylation-based imprinting at specific loci.

Perturbations of both global and gene-specific patterns of cytosine methylation are commonly observed in cancer while Kim et al [8] showed that histone deacetylation is an important feature of nuclear reprogramming in oocytes during meiosis.

Recent studies have revealed that there is an array of different pathways that cooperates with one another in order to bestow proper epigenetic regulation by DNA methylation. Future studies will be needed to further clarify the certain mechanism pathways such as DNA binding proteins, DNA repair and noncoding RNAs serve in order to regulate DNA methylation to suppress differentiation and sustain self-renewal in somatic stem cells in the epidermis and other tissues. Addressing these questions will help extend insight into these recent findings for a central role in epigenetic regulators of DNA methylation in controlling embryonic stem cell differentiation [9].

The initial version of this document was written as part of an epigenetics class at the University of Texas at Austin.